I BIOLOGICAL CONTROL OF ’ SOYBEAN ROOT ROT CAUSED BY THIELAVIOPSIS BASICOLA (BERK. 8: BR.) FERR. Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY MICHAEL ABDI ZAKARIA 1975 THESIS L I B R A R Y Mldsba'l State ‘nivcrsicy :'_._ -. u ‘ i 1.x“. r _ .. a...;.l’f'~‘“‘t' F“ ll—ii." (IL I a. r ‘ . . alfiilugey 3‘ , 'IIUIIB 8 SONS' 800K Rmfgyr'uig % i um- . l‘ . ucnnm “g.“uvrlr II I h"-mmfim *wm‘mfi‘." 4...; L c..." ,awmar'l'al‘ 5.5" ~ I p__._._._‘ "LJLHL T Whacg--3.,.gr_-‘ 2 ' ‘2 -'.'~'m — - m krl‘h »-—— u . ‘ ’1 A. ’1 '3 1L1 ant-3" JW i i 13.2".” :__". IIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 193 10710 1465 2 ABSTRACT BIOLOGICAL CONTROL OF SOYBEAN ROOT ROT CAUSED BY THIELAVIOPSIS_BASICOLA (BERK. & BR.) FERR. By Michael Abdi Zakaria The effects of temperatures ranging from 1650-28°C and inoculum concentrations ranging from 0.31 9-5 9 of inoculum/kg soil were tested on the development of soybean root rot caused by Thielaviopr sis basicola. Disease development was enhanced both by lower tempera- tures and higher inoculum concentrations, but temperature showed a much greater effect than inoculum concentration. I A bilayer method was used for the collection of antagonists. In this method, soybean roots with closely adhering soil were mixed with selective agar media for bacteria, actinomycetes, and fungi as the bottom layer. PDA seeded with I, basicola endoconidia was used as the top layer. Rhizospheres of soybean grown in soil amended with either chitin, ground dried alfalfa, or non-amended were used as sources of antagonists. Several bacterial,.actinomycete, and fungal antagonists were also collected by random selection directly from soybean rhizo- sphere soil and from lysing Fusarium hyphae. In paired cultures. antagonism involving overgrowth of I, basicola was shown by fungal isolates; and production of inhibition zones by bacterial, actinomycete, and several fungal isolates. In many Michael Abdi Zakaria instances, fungal overgrowth of I, basicola-resulted in loss of viability of the pathogen. The inhibition zones were associated with either antibiotic production, inhibition of endoconidial germination, bursting of endoconidia. lysis of hyphae and endoconidia, or combina- tions of the four characters. Inhibition zones from antibiotic produc- tion were smaller as the nutrient content of the agar medium decreased. Larger inhibition zones were produced on PDA than on seed exudate agar, and some isolates producing inhibition zones on PDA failed to do so on seed exudate agar. Promising results were obtained from biological control exper- iments by applying antagonists to seeds sown in steamed soil or natural Conover loam. Three bacteria, five actinomycetes, and four fungi showed significant reduction in disease severity in steamed soil mix; and three bacteria, no actinomycetes. and one fungus. in Conover loam. 0f the three bacteria. one reduced disease significantly in both soils. How- ever. antagonists isolated directly from the rhizosphere through random selection gave greater success in biological control than those isolated by the bilayer method or from the lysed Fusarium hyphae. Six of twelve 'antagonists selected randomly from the rhizosphere significantly reduced disease severity in steamed soil, and four did so in Conover loam; success in biological control obtained by antagonists isolated otherwise was only 14%. Greater success in biological control was also shown by the antagonists which did not produce diffusible antibiotics (34%). as compared to those that did (l4%). Isolates which produced distinct inhibition zones in paired cultures on seed exudate agar (29%) showed greater success than those which produced none or indistinct inhibition Michael Abdi Zakaria zones (l7%) in steamed soil mix. However, success in Conover loam was about equal for the two groups, i.e.. 6% and 8%, respectively. Other characteristics of antagonism. either individually or in combinations, were not consistently associated with success in bio- logical control. An approach to evaluation of antagonists using water agar seeded with I, basicola endoconidia as the growth medium for the coated soybean seeds was tested. Reduction in disease severity obtained using this method showed some correlation with the success in biological control in soil. This method has a short test period and it provides a transparent medium, making possible observations of disease develop- 'ment and possibly of the interactions of the antagonists with the pathogen in the rhizosphere. BIOLOGICAL CONTROL OF SOYBEAN ROOT ROT CAUSED BY THIELAVIOPSIS BASICOLA (BERK. & BR.) FERR. By Michael Abdi Zakaria A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1975 To my wife and parents ACKNOWLEDGMENTS I would like to express my deepest appreciation to my major professor, Dr. J. L. Lockwood, for his guidance and constant encourage- ment. and especially for his patience and assistance in the preparation of this manuscript. Thanks are also conveyed to Dr. w. G. Fields and Dr. A. w. Saettler for advice and for critical evaluation of the manuscript. I would also thank Dr. B. Sneh for his useful suggestions and comments, and Mr. D. M. Weller for his assistance in the identification of several bacteria. To all my laboratory-mates, for their cooperation, I express my gratitude. TABLE OF CONTENTS LIST OF TABLES ....................... LIST OF FIGURES ...................... INTRODUCTION ........................ LITERATURE REVIEW . . . . . . . . ........ . . . . . The pathogen. its taxonomic status and epidemiology. Antagonism . . . . . . . . . . . . . . . . . . . . I 2 Biological control ................ . . . . MATERIALS AND METHODS ................... Preparation of inocula . . .-. ...... ‘. . . . Effect of soil temperature and inoculum concentration on disease severity ...... . . . ..... . . . . Sources of antagonists . ............... -. Evaluation of antagonists ................ Preservation of isolates . . . . . . .......... Antagonism on agar plates ................ Antibiotic production and nutrient deprivation on diluted PDA . . ............ Assays of biological control .of T. basicola of soybean . . ..................... An alternative method for evaluating antagonists for biological control ................ RESULTS ....... . ...... . ..... . . . . . . Effect of soil temperature and inoculum concentration on disease severity . . . . . . . . . . ....... Collection of antagonists . . . . . . . . . ...... Analysis of antagonism on agar plates . ......... Antibiotic production and nutrient deprivation on diluted PDA ................. Biological control experiments with soybean root rot caused by I_. basicola . ............. Effect of coated soybean seeds on root rot development in water agar .............. iv Page vi ix A 00100 0..) 24 27 39 39 48 Page DISCUSSION .......................... 54 LITERATURE CITED ....................... 64 LIST OF TABLES Table Page 1. Production of inhibition zones, antibiotic produc— tion, characteristics of the inhibition as determined microscopically, and growth habit of bacterial isolates three days after spraying agar plates containing individual isolates with a suspension of Thielaviopsis basicola endo- conidia . . . . . . . . . . . . . . . . . . . . . . . 28 2. Reproduction of inhibition zones by seed exudate agar taken from inhibition zones of the bacte- rial isolates obtained by the bilayer method, and viability of Thielaviopsis basicola endo- conidia in the reproducedinhibition zones ..... 30 3. Inhibition zone production and growth of bacterial antagonists applied with seeds to carrot- extract agar seeded with Thielaviopsis basicola endoconidia. Seeds were either left on t e agar for five days. or were removed after one minute . . . . . . . . . . . . . . . . . . . . . . . 31 4. Production of inhibition zones by actinomycetes, antibiotic production, and characteristics of the inhibition as determined microscopically three days after spraying agar plates contain- ing individual isolates with a suspension of Thielaviopsis basicola endoconidia ......... 33 5. Production of inhibition zones by filter.paper discs impregnated with culture filtrates of actinomycete isolates, and viability of Thiela- viopsis basicola endoconidia in the inhibition zones surrounding the filter paper discs . . . . . . 34 6. Production of inhibition zones by fungi isolated by the bilayer method, antibiotic production, and characteristics of the inhibition as deter- mined microscopically three days after spraying agar plates containing individual isolates with a suspension of Thielaviopsis basicola endoconidia . 36 vi Table IO. II. 12. 13. I4. 15. Page Production of inhibition zones by filter paper discs impregnated with culture filtrates of fungal isolates, and viability of Thielaviopsis basicola in fifteen-day-old overgrown cdltures . . . . 37 Effect of various dilutions of PDA on formation of inhibition zones against Thielaviopsis basi- cola by nutrient-depriving calonies OfTactino— mycetes . . . . . . . . . . . . . . ..... . . . . 41 Effect of bacterial and fungal antagonists applied to soybean seeds on root rot caused by Thiela- vio sis basicola. Seeds were sown in a previously steamed soil mix infested with endoconidia and chlamydospores of the pathogen . . . . . . . ..... 45 Effect of bacterial and fungal antagonists applied to soybean seeds on root rot caused by Thiela- viopsis basicola. Seeds were sown in a previously steamed soil mix infested with endoconidia and chlamydospores of the pathogen . . . . . . ...... 46 Effect of actinomycete and fungal antagonists applied to soybean seeds on root rot caused by Thielaviop- sis basicola. Seeds were sown in a previously steamed‘soil mix infested with endoconidia and chlamydospores of the pathogen . . . . . . . . . . . . 47 Effect of bacterial, actinomycete, and fungal anta- gonists applied to soybean seeds on root rot caused by Thielaviopsis basicola. Seeds were sown in a Conover Team-white silica sand mix infested with endoconidia and chlamydospores of the pathogen .......... . . . ....... 49 Several morphological and physiological character- istics of three bacterial antagonists giving significant biological control of soybean root rot caused by Thielaviopsis basicola in Conover loam ......... . . . ....... . . . . . . 50' Effect of soybean seeds coated with antagonists on root rot development in water agar seeded with endoconidia of Thielaviopsis basicola . . . ..... 53 Results of laboratory observation of bacterial antagonists as related to their performance in the biological control experiments with soybean root rot caused by Thielaviopsis basicola . . . . . . 56 vii Table Page l6. Results of laboratory observation of actinomycete antagonists as related to their performance in the biological control experiments with soybean root rot caused by Thielaviopsis basicola . . . . . . 57 17. Results of laboratory observation of fungal anta- gonists as related to their performance in the biological control experiments with soybean root rot caused by Thielaviopsis basicola ...... 58 viii Figure LIST OF FIGURES Disease index scale used for estimating severity of Thielaviopsis basicola root rot in soybean plants grown individually in test tubes. Disease was rated on a scale of 0-6, with 0 indicating no disease and 6, maximum disease severity O O ........ O O O O O O O O O I O 0 Effect of different soil temperatures and inoculum concentrations on root disease severity of soy- bean cultivar 'Harosoy-63' caused by Thiela- viopsis basicola. Each bar represents the mean of four replicates with 4-5 measurements in each replicate . . . . . .2. . . . . . . . . . . . . . . Viability of overgrown Thielaviopsis basicola in paired cultures with six different fungal anta- gonists as shown by the number of colonies arising on a selective medium. A) Rhizo us sp., B) an asporogenous deuteromycete, C E BI Fusarium spp., E & F) Penicillium spp. Darkest colonies are I, basicola, some of which are indicated by arrows Effect of various dilutions of PDA on production of diffusible antibiotics, as expressed by the width of the inhibition zones produced. b2 and b9 were bacteria, f9 was a fungus (Penicillium sp.), and Ll was a Streptomyces ...... . . . . Reduction in severity of soybean root rot caused by Thielaviopsis basicola, by antagonists applied to the seeds. The seeds were sown in steamed soil mix infested with the pathogen. Left, control; center, seed-coating with bac- terial isolate sb2; right, seed-coating with fungal isolate Tl3 (Trichoderma harzianum) ..... ix Page 22 25 38 4O 43 Figure Page 6. Reduction in severity of soybean root rot caused by Thielaviopsis basicola, by antagonists applied to the seeds. The seeds were sown in steamed soil mix infested with the pathogen. Left. control; center, seed-coating with bac- terial isolate b9; right, seed-coating with bacterial isolate sb4 . . . . . . . . . . . ..... 43 7. Reduction in severity of soybean root rot caused by Thielaviopsis basicola, by antagonists applied to the seeds. The seeds were sown in water agar seeded with the pathogen. Left, control; center. seed-coating with bacterial isolate b9; right, seed-coating with fungal isolate T13 (Trichoderma harzianum) . . . ...... 52 INTRODUCTION Thielaviopsis basicola (Berk. & Br.) Ferr. is a common pathogen of many crops, e.g., tobacco, cotton, citrus, but it was first reported causing root rot of soybeans in Michigan in 1970 (49). This disease occurs widespread throughout the eastern portion of the lower half of the lower peninsula. Since then, work has been done at Michigan State University on the epidemiology of the disease. Lower temperatures have been found to favor disease development, while temperatures above 24°C markedly decreased disease severity (Chen and Lockwood, unpublished results). Soybean plants of ages ranging from 0-21 days have been shown to be susceptible to severe infection provided low temperature 'requirements for disease development are met (Zakaria and Lockwood, unpublished results). Under low temperatures, disease can be produced on plants grown in soil infested with an inoculum concentration as low as l02 propagules per gram of soil (Maduewesi and Lockwood, unpublished results). Although different soybean varieties showed different degrees of response to the pathogen, none proved to be immune. Work has been done on the influence of antagonistic fungi on I, basicola attacking tobacco (73). Although the fungi maintained their antagonistic properties in laboratory experiments as well as in sterilized soil, they were not effective in unsterilized soil. With increasing knowledge of the manipulation of soil envi- ronments and their microorganisms, this study was undertaken to 1 investigate the possibilities of performing biological control of I, basicola root rot of soybeans. LITERATURE REVIEW The pathogen, its taxonomnc status and epidemiology According to the Saccardo system of classification, the genus Thielaviopsis belongs to the family Dematiaceae, of the order Moniliales (8.30). No perfect stage of this fungus has so far been found. The conidia are rod-shaped, one-celled, hyaline, and borne endogenously from 'sporogenous cells, the phialides. Besides the hyaline conidia, this fungus also produces dark chlamydospores (aleuriospores), each consisting of several cells arising from transverse septation. According to the Hughes-Tubaki-Barron system of classification, this fungus is grouped in the series Phialosporae (8,9). Thielaviopsis basicola is the only species of the genus recorded from soil (9). The fungus is known as an aggressive pathogen causing root rot on a large host range, although variations in virulence exist among isolates from different hosts (45,46). This fungus has also been found associated with root necrosis of Phytgphthora-resistant soybeans (68). I, basicola survives in soil mainly as chlamydospores which remain viable over a long period of time at low moisture and low tem- peratures (61,76). Under similar soil conditions a considerable number of endoconidia were found to survive after eight months, but a great reduction in number occurred in moist soil incubated at higher temperatures. In moist soil the chlamydospores break up into segments each of which is capable of causing infection (44.61.62). In the absence of host plants, germinability of chlamydospores declined rapidly after 20-45 days at 40-45% of moisture-holding capacity (MHC). No decline in germinability was observed at 15% of MHC with a temperature maintained at 20°C. In air-dry soil germinability declined to 90% at '10°C and 18°C and to 30% at 34°C after three months (61). Clough and Patrick (20) found that loss in chlamydospore viability in soil was associated with naturally occurring perforations in the spore walls. Bacteria were often seen in association with the perforations, but a causal relationship has not yet been established. From laboratory studies, it was found that chlamydospore germination is stimulated by glucose, sucrose, mannose, and especially 'by root exudates (52). Some chlamydospores, however, were capable of germinating in the absence of exogenous nutrient (44). In nature, germination mainly occurs in the immediate vicinity of the host (45). During infection, the disease progresses as the hyphae grow within the host tissue and upon the root surface, giving rise to new infection loci (45). On soybeans, black to brown necrotic symptoms are produced on the cortex of the below-ground portion of the hypocotyl and of the tap and fibrous roots (49). Secondary inocula are produced after the development of root rot symptoms, resulting in a marked increase in the number of propagules in the rhizosphere (10,62). In general, disease development for high temperature plants like tobaCco, cotton, and Navy beans is favored by a temperature range of 18°C-23°C; temperatures above 26°C markedly lower the disease severity (52,73). However, disease development in peas, a cool temperature plant, is favored by higher temperatures (46). Most severe disease in peas has been obtained at a temperature of 28°C. The relationship between disease severity and temperature, therefore, appears to depend on the optimum temperature for the host plants. Disease development is also favored by pH values higher than 5.6, although on cotton severe disease can be obtained at a pH value as low as 4.0 (52,73). Antagonism Antagonism among microorganisms may be attributed to anti- 'biosis, competition, or exploitation (5,31). 0n agar plates antagonism ‘may be exhibited by the formation of inhibition zones. Lytic zones have been observed on agar plates both by antibiotic-producing and nutrient-depriving-actinomycetes (33).‘ Antagonism through antibiosis requires that the organisms producing the toxic substances have adequate nutrients (l3). Produc- tion of antibiotics and other staling products may enhance the com- petitive ability of the organisms producing them (16,24). In the case of competition, energy-yielding nutrient seems most likely to be of limited supply (19). That a sizable proportion of the soil microorganisms should be dormant at any given time reflects the lack of suitable and available energy—yielding nutrient. The importance of exogenous and endogenous nutrients on the germination of nutrient-dependent and nutrient-independent spores in soil has been discussed in detail by Lockwood and his co-workers (34,39,47,48,75,78). Failure of germination in soil can be attributed to lack of essential exogenous nutrients or rapid loss of essential endogenous nutrients by competing microorganisms. Rapid germination followed by rapid growth prOvides advantages in the process of competition (23,75). Spores with long germination times may not produce germ tubes if the nutrients in their surroundings are rapidly exhausted due to the competitive activities of other microorganisms. Other attributes suggested for a successful competitor include the capability of producing toxic sub- 'stances, tolerance to toxic substances produced by other microorganisms, and possession of appropriate enzyme systems for substrate decomposi- tion (24,28). Antagonism in the form of exploitation involves predation and hyperparasitism (5). There are two means suggested by which hyper- parasitism occurs (7,12). In necrotrophic parasitism, toxic substances may be produced while the parasite is making contact with its host. ' The parasite will thus kill the host and utilize the released nutrients. In biotrophic parasitism the parasite obtains nutrients from living host cells without previously killing them. The latter form of para— sitism is most often found in nature. Phenomena of haustorial forma- tion, internal growth of parasitic hyphae, and coiling of parasitic hyphae around the host have been observed ifl.!i££2.(7): but their occurrence in soils is still questionable. Biological control Biological control of soil-borne plant pathogens could be achieved if antagonism in favor of the non-pathogens could be maintained in the soil (5). One important mechanism of such antagonism involves soil fungistasis which is of widespread occurrence in natural soil. It is characterized by a non-specific inhibition affecting almost all species tested, coexistence with microbial activities . and can be ’nullified by nutrients (47). Germination of spores of fungi in soil is generally restricted to the nutrient-rich environments of plant rhizospheres and plant residues. or follows artificial amendment of soil with energy-yielding nutrients (78). Ko (38) successfully utilized the soil fungistatic phenomena in the biological control of seedling root rot of papaya caused by Phytophthora palmivora. He~used pathogen-free soil to replace a core ‘of pathogen-containing soil in the field as a growth medium for the papaya during the susceptible stage. The pathogen was excluded due to the fungistatic effect of the pathogen-free soil. Papavizas and his co-workers (1.59.60) used organic amend- ments such as alfalfa hay. corn stover, and oat straw, to stimulate germination of I, basicola chlamydospores in soil. However, germina- tion was followed by lysis due to antagonistic activities of soil microorganisms which were also stimulated by the amendments. They also showed that the fungistatic effect of the soil was increased after the amendments. The importance of plant exudates in relation to diseases has widely been studied. Plant exudates directly affect the pathogens by inducing their germination and by contributing to their nutritional status prior to infection (ll.26.41.69.7l). For example. Rai and Strobel (63) observed that attraction of zoospores of Aphanomyces cochlioides to roots of sugar beets. and germination was stimulated by different components of root exudates. Kraft (42) related resistance of peas to Fusarium and Pythium root rots to qualitative differences in the seedling exudates. 0n the contrary. Keeling (35) found no qualitative differences in the exudates of soybean seeds of resistant and susceptible cultivars to Pythium ultimum and E, debaryanum. but more than twice the quantity of soluble carbohydrates was exuded from seeds of the susceptible cultivar. Plant pathogens are affected indirectly through competition and antibiosis by the epiphytic microorganisms whose activities also "are mediated by plant exudates (21.25.29.43.70,7l). Germlings of Fusarium solani f. phaseoli lysed due to microbial activity in the nutrient-rich environment around exuding bean seeds, but lysis did not occur and hyphae continued to develop around the hypocotyl where exuda- tion was less (21). PCNB-tolerant microbes have been shown to increase more rapidly in nutrient-amended soil containing PCNB than in soil without PCNB (25). This reduced competition by PCNB-sensitive micro- Organisms was suspected to be a mechanism for PCNB-induced disease accentuation by tolerant fungi. Plant pathogens can establish themselves in the rhizospheres, particularly when favored by a host as a selective substrate (5). However. if a high number of microorganisms were for some reason already growing actively when an exuding plant part first appeared, the nutrient liberated might quickly be absorbed and a steep gradient consequently maintained from the outset, resulting in fewer germinated pathogenic propagules. Successful performance in biological control using resident antagonists has been achieved by several workers. Fokkema and Lorbeer (27) used saprophytic mycoflora of onion leaves to reduce infection in Alternaria porri. A-Fusarium species isolated from colonized tissues ‘below a pruning wound was found capable of controlling die back of apricot caused by Eutypa_armeniacae-(l7).o Macroconidial suspensions of the Fusarium applied to wounds immediately after pruning reduced 'infection by the pathogen. Rishbeth (65,66) used Peniophora gigantea, ~a'natural stump colonizer to prevent invasion of freshly cut pine 'stumps by Fomes annosus. The~antagonist was also capable of replaCing. 'to some extent. the pathogen in already colonized stumps. In another 'approach to biological control, planting grass leys prior to wheat 'stimulated the development of Phialophora radicicola. a non-pathogenic ‘ectotrophic root parasite of graSses (22). This fungus has a growth "habit similar to that of the pathogen. Gaeumannomyges graminis. and was found capable of reducing infection by the pathogen. Marx and Davey (51) found that fully formed mycorrhizal roots ~of‘pine seedlings were resistant to infection by zoospores of Phytoph- thora cinnamomi. Although the zoospores were attracted to excised root tips. infection did not occur on mycorrhizal roots even with artificially lexposed cortical tissue. Marx (50) suggested several possible mechanisms for resistance. viz., nutrient deprivation. physical barrier. stimula- tion of rhizosphere microbial growth. and production of inhibitors by the plant and antibiotics by the mycorrhizal fungus. Similar mechanisms were suggested by Stack and Sinclair (74) from their work on protection of Douglas-fir seedlings against Fusarium root rot by mycorrhizal fungi. IO Antagonism through the production of antibiotics requires ‘that sources of nutrient such as plant residues. laimosphere, and rhizo- sphere be available (6.77). Although antibiotics can rapidly be inacti- 'vated by soil particles or microbial degradation, they may be important 'for limited periods while the environment favors their production (15). For example. production of bulbiformin by Bacillus subtilis-has been observed on seed coats, spermatospheres. and rhizospheres of bacterized pigeon-pea seeds in sterile and unsterile soil (72). Production was enhanced when molasses and groundnut cake were applied to the seeds ’ with the bacteria, and this resulted in reduction of wilt by Fusarium ugum, Biological control using rhizosphere antagonists applied 'through seed-coating and root-dipping has been carried out with some 'success (2.36.55.56.58). Increase in yield derived from applications of antagonistic microorganisms has also been claimed (40.53.54). “Atkinson et_gI, (3.4). however. found that although antagonists were ‘more prevalent in the rhizosphere and in the laimosphere of resistant varieties of wheat. seed bacterization with the antagonists did not increase resistance of the susceptible varieties to Cochliobolus sativus. Moreover. one breeding line, although susceptible. was also shown to harbor antagonists in a comparable number to those of the resistant line. Thus, antagonists and resistance were non-related in their system. Antagonism in agar media does not necessarily predict anta- gonistic activity in soil (6.36). Since a very low percentage of antagonists isolated from soil proved effective under commercial ll conditions. Broadbent e§_gl; (14) suggested that large scale screening methods are essential. However. screening for antagonists may be made 'more efficient using better approaches in the evaluation technique (32. 57). accompanied by better specifications in environmental requirements such as substrate (15.17.36) and temperature (17.18) suitability. MATERIALS AND METHODS Preparation of inocula Thielaviopsis basicola isolate 157 (from Monroe Co.. Mich.) was used throughout the experiments. The fungus was maintained as a stock culture on potato dextrose agar (PDA) slants at 4°C. For produc- ‘tion of inocula, the fungus was grown either on soybean-sand medium, in carrot-extract broth. or on PDA slants. The soybean-sand medium was prepared by mixing 200 9 ground 'soybean seeds (prepared by grinding 200 9 dried soybean seeds in a 'Waring Blendor for about five minutes), 1000 9 white silica sand. and 250 ml distilled water; followed by sixty-minute steaming. The steamed mixture was broken up with a fork before putting it into Erlenmeyer flasks for sterilization. I, basicola was grown in the medium for 4-6 weeks at 24°C before use. Carrot-extract broth was prepared by autoclaving 200 g carrot root slices in 1000 ml distilled water for fifteen minutes. The mixture was then passed through four layers of cheese cloth and the cheese cloth containing the carrot was squeezed until most of the liquid was extracted. The extract was resterilized in 250 ml Erlenmeyer flasks which. after cooling. were inoculated with I, basicola. The flasks were incubated at 24°C for 2-3 weeks. The liquid cultures were then homogenized in a Servall Omnimixer for ten minutes and the homogenates were washed by repeated centrifugation. The washed homogenates were resuspended in 12 13 distilled water and the propagule (mixture of endoconidia and chlamydospores) concentration was measured using a hemacytometer. An ‘intact chlamydospore chain, a single segment; or a short chain of several segments. each was counted as a single propagule. Slant cultures were prepared by inoculating I, basicola on PDA slants in test tubes, followed by one week's incubation at 24°C. Endoconidial suspension was prepared by adding 5 ml distilled water into each test tube, followed by shaking vigorously using a Vortex Jr. mixer for one minute. Inoculum concentration was measured using a hemacytometer. This endoconidial suspension was used for work on anta- gonism on agar plates. Unless otherwise stated. the final concentration was adjusted to 106 endoconidia/m1 and 0.05 ml was applied to each plate using a De Vilbiss atomizer. 'Effect of soil temperature and ihocuTUm concentration on disease severity An experiment was carried out in the greenhouse to test the effect of different temperatures and inoculum concentrations on disease severity. Controlled temperature water baths at 16°C. 20°C. 24°C. and 28°C were used. The inoculum concentrations used were5 g, 2.5 g. 1.259% 0.63 g. and 0.31 g of soybean-sand culture per kg of steamed soil mix (compost. peat. coarse sand; 1:1:1 v/v/v). Uninfested steamed soil mix was used as control. Four to five 'Harosoy-63' soybean seeds were planted in each of four plastic pots for each treatment. The pots were incubated for two days on a greenhouse bench and then transferred 14 'to the water baths for three week's additional incubation before disease severity was assessed. Watering was done daily by maintaining 'constant weight in each treatment: 1160 g/pot for 16°C. 1180 g/pot for 20°C. 1190 g/pot for 24°C. and 1200 g/pot for 28°C. Disease in ‘each plant was rated on a scale of 0-5; 0 being disease-free. l slightly diseased. 2 slightly to moderately diseased. 3 moderately diseased. 4 moderately to severly diseased, and 5 severely diseased. Sources of antagonists Conover loam (46) amended either with 1% (w/w) chitin or 1% (w/w) ground dried alfalfa hay. or non-amended was used as sources of ‘antagonists.. Four soybean seeds were planted in single plastic pots ‘containing the amended or non-amended soil and incubated in a water- 'tank at 19:1°C in a greenhouse for fifteen days. The seedlings were then uprooted. the roots washed with running tap water to remove adher- ‘ing soil and ground in a mortar. The ground roots were made into 50 ml 'suspensions which were further diluted with distilled water from 10"2 to 10"3 for plating purposes. Serial dilutions were also prepared from rhizospheres of soy- 'beans grown in Conover loam in the greenhouse and from mycelial mats of Fusarium solani placed on small pieces of nylon net and buried in soil for two days. These were also then used as sources of antagonists. Evaluation-of antagonists Evaluation of antagonists from the root suspensions was car- ried out using a bilayer technique according to the method of Henis 15 gt_§I, (32). Three different selective media were used for the basal layer. For actinomycetes the medium contained 4 g colloidal chitin. 20 g Difco agar. and l000 ml distilled water (pH 7), and will be referred to as chitin agar. For bacteria the medium contained 100 ml soil extract. 1 9 glucose. 0.5 g KZHP04. 20 g Difco agar, 900 ml dis- tilled water, and 0.05 g pentachloronitrobenzene (PCNB); the pH was adjusted to 6.8-7.0—-this medium will be referred to as soil-extract agar. For fungi. PDA with 0.5 ml TMN detergent (Union Carbide Co.) and 0.25 g chloramphenicol (added after autoclaving) was prepared--this medium will be referred to as PDA-chloramphenicol. The serial dilu- 'tions from the root suspensions were plated using these selective media. - The plates were incubated at 24°C for 2-4 days before applying the top layer. PDA seeded with I, basicola endoconidia was prepared in Petri 'plates and incubated for l-2 days. This agar layer was transferred to a sterilized cellophane sheet and then was aseptically transferred onto 'the bottom layer. These bilayer cultures were incubated for two or more additional days and colonies showing production of inhibition I ‘zones and/or overgrowth of I, basicola were‘isolated. Antagonistic bacteria and fungi were transferred to PDA and actinomycetes to chitin agar. Similar selective media were used for the isolation of bac- teria. actinomycetes. and fungi from soybean rhizospheres and the Fusarium mycelial mats. Several of these isolates were chosen for evaluation of antagonism, using the paired culture technique according 16 ‘to the method of Hsu and Lockwood (33). Antagonists were isolated and transferred to the same media. Preservation of isolates~ Isolates were preserved either on agar slants or in glycerol solution. Fungal isolates were grown on PDA slants for a few days in 'screw-capped test tubes at 24°C before being kept at 4°’C. Actinomycete 'isolates were preserved in a similar manner. except that yeast-extract agar (1000 ml distilled water: 2 g yeast extract and 20 g Difco agar) 'slants were used. Bacterial isolates were grown in 5 ml nutrient broth for several days before 2 ml of 80% glycerol was added. These cultures were maintained in glass vials and kept at 42°C. Antagonism on agar plates The various isolates were tested for antagonism to I, basicola ‘on PDA and on seed exudate agar. The latter medium was prepared by _ soaking 200 g surface-sterilized (fifteen minutes in l000 ppm NaOCl) soybean seeds in 600 ml distilled water for twenty-four hours. then passing the exudate solution successively through Whatman No. 5 and No. 50 filter papers.‘ Distilled water was added to the filtrate to make a final volume of 500 ml, into which was then added 10 g Difco agar before autoclaving. A 1. Production and assays of inhibition zones. Isolates were inocu- lated individually at the centers of PDA and seed exudate agar plates, and incubated for two days before application of an endoconidial sus- pension of I, basicola using a De Vilbiss atomizer. After another two 17 days' incubation the plates were examined for zones of inhibition in the otherwise uniform growth of I, basicola. Inhibition zones were measured from the margins of the antagonistic colonies to the margins of the inhibition zones. Microscopic observation of the inhibition zones was carried out one. two and three days after spraying by directly observing the plates under the microscope. Occurrence of poor germination of I, basicola endoconidia. poor growth. bursting of endoconidia. lysis of hyphae, and parasitism was noted. Detection of antibiotic production and nutrient deprivation 'in the agar was carried out by transferring agar discs (7 mm in dia- meter). cut with a cork borer. from the inhibition zones to the surface 'of PDA plates. For those isolates producing smaller inhibition zones, agar squares (5 mm x 5 mm) were cut with a spatula and transferred. After application of an endoconidial suspension of I, basicola, the 'plates were incubated at 24°C for two days and the presence of new 'inhibition zones was then observed to see if diffusible inhibitory substances were produced. Inhibition zones were measured from the margins of the agar discs or squares to the margins of the inhibition zones. For those isolates producing very small inhibition zones in paired cultures and those showing overgrowth of I, basicola. another method for detection of antibiotic production was used. The isolates were grown in seed exudate broth for one week when culture filtrates were tested for the presence of antibiotics. Two ml of each culture filtrate was passed through a Nuclepore filter (0.22 um; 10 mm diameter) 18 in a Swinnex adapter attached to a hydrodermic syringe. Three drops of each culture filtrate were applied to sterilized filter paper discs (13 mm diameter) and the discs were placed on PDA plates. The plates were sprayed with an endoconidial suspension of I, basicola and forma- tion of inhibition zones was.observed after two days. Inhibition zones were measured from the margins of the filter paper discs to the margins of the inhibition zones. 2. Viability tests. Viability of I, basicola from the inhibition zones produced by the transferred agar discs and by the filter paper discs ‘impregnated with the culture filtrates was analyzed. Agar discs (7 mm diameter) or agar squares (5 mm x 5 mm. for those isolates producing "smaller inhibition zones) were taken from the inhibition zones. inverted. and placed on fresh PDA plates. ’The plates were incubated at 24°C for one week and growth of I, basicola from-the transferred agar discs or squares was observed. Viability of I, basicola in the areas overgrown by fungal isolates was also determined. Seed exudate agar in Petri plates was 'seeded with I, basicola endoconidia and incubated for two days at 24°C. Individual fungal isolates were then inoculated at the centers of the plates. The plates were incubated for fifteen days before discs (7 mm diameter) taken from the overgrown areas next to the inoculation points were ground in 5 ml distilled water for one minute using a glass tissue homogenizer. Each homogenate was made into a final volume of 10 ml by adding distilled water. and 0.5 ml was then plated with 5 ml of a selective medium for I, basicola (Maduewesi et al.. unpublished results). 19 Colonies of I, basicola arising from the plates were counted after 6-7 days. Control plates were prepared from similar suspensions. but agar discs from pure I, basicola plates were used instead. Antibiotic production and nutrient deprivation on diluted PDA PDA either undiluted or diluted to l/2. 1/4, l/8, l/l6. l/64. and l/128 normal strength was used in the assay. Isolates known to produce inhibition zones through antibiosis and nutrient deprivation were individually streaked at the centers of PDA plates. Agar discs (7 mm diameter) were taken from the margins of antibiotic-producing colonies incubated at 24°C for 3-4 days and were transferred onto fresh undiluted PDA plates. The plates were then sprayed with an endoconidial suspension of I, basicola. Inhibition zones produced were measured two days after spraying. Isolates produc- ‘ing inhibition zones through nutrient deprivation were individually streaked on PDA plates prepared at various dilution from 1/1 to 1/128. After 3-4 days the plates were sprayed with a suspension of I, basicola endoconidia. Inhibition zones were measured three days after spraying. Measurement of inhibition zones was done as previously described. Assays of biological control of T} baSicola of soybean .1. Preparation of cell and spore-suspension. Bacterial and fungal isolates were grown on PDA slants in test tubes for about ten days before 3 ml of 0.1% water agar was added to each tube. The tubes were vigorously shaken using a Vortex Jr. mixer for one minute. and the 20 resulting suspensions were collected'in glass vials. Spore suspensions of actinomycetes were prepared in a similar manner except that yeast- dextrose agar slants and 0.1% chitin agar were used. Yeast-dextrose agar contained. per liter: yeast extract 10 g, dextrose 10 g. CaCO3 2.5 g. and Difco agar 20 g. Isolates which did not produce abundant spores were scraped from the slants and homogenized in a glass tissue homogenizer for two minutes with the same amount of 0.1% agar solution. 2. Seed coating. Homogeneous soybean seeds (cultivar 'Hark') were 'selected. Sixteen seeds were soaked in the cell or spore suspensions for three minutes before placing them individually in soil in polysty- rene test tubes 10 cm long x 1.6 cm diameter (Maduewesi and Lockwood. unpublished results). Control seeds were similarly soaked in 0.1% water agar or 0.1% chitin agar. 3. Soil preparation and planting. Steamed soil mix was passed through a 2 mm sieve before infesting it with I, basicola spore (mixture of endoconidia and chlamydospores) suspension. Infestation was done by spraying the spore suspension onto the soil in a drum rotating on an electric-powered rotary mixer. Unless otherwise stated. the final inoculum concentration was 103-104 propagules per 9 soil. Natural Conover loam was similarly prepared, except that 20% (w/w) of white silica sand was mixed with the soil before infestation and the final 4-105 propagules per 9 soil. inoculum concentration was adjusted to 10 Uninfested soils were used for comparison. The soil was wetted to about 50% MHC before transferring about l5 9 to each polystyrene test tube. To compact the soil. each tube was gently tapped three times on a hard surface. Coated seeds 21 were placed on the top of the soil columns and enough soil to cover the seeds was added. The tubes were incubated at 25°C under continuous fluorescent light (8.600 lux) for three days. during which time the seeds germinated. The seedlings were further incubated in a growth chamber maintained at 20°C. with a photoperiod of fifteen hours per day and a light intensity of 10,200 lux. Disease assessment was carried out after fifteen days. - *4. Disease assessment. Fifteen days after planting. the seedlings were removed from the polystyrene tubes. The roots were then washed with running tap water. Disease was assessed on a scale of 0—6. 0 indicates a disease-free state of the root system; 1. lesions present on up to *25% of the root system but no girdling of tap root; 2, lesions present on up to 50% of the root system but no girdling of the tap root; 3, lesions on more than 50% of the root system or a girdled lesion of up to l cm in length; 4, girdling as in 3 but with additional lesions on up to 25% of the root system. or a girdled lesion of 1.1-1.5 cm in length; 5, a girdled lesion of l.l-l.5 cm in length with additional lesions on up to 25% of the root system. or a girdled lesion of 1.6-2.0 cm in length; and 6. a girdled lesion of 1.6-2.0 cm in length with additional lesions covering up to 25% of the root system. or a girdled lesion of more than 2 cm in length (Figure 1). An alternatige method for evajuating; antagonists for biological control' Soybean seeds (cultivar 'Hark‘) were surface—sterilized by soaking them in 1000 ppm NaOCl for fifteen minutes. After rinsing three 22 Figure 1. Disease index scale used for estimating severity of Thielaviopsis basicola root rot in soybean plants grown individually in test'tubes. Disease was rated on a scale of 0-6. with 0 indicating no disease and 6. maximum disease severity. 23 times with sterile distilled water, the seeds were then coated with aqueous suspensions of the antagonists. Glass test tubes (15 cm x 2.3 cm) were one-third filled (20 'ml) with 0.8% water agar and maintained at 45°C in a water bath._ One ml of an endoconidial suspension of I, basicola was vigorously mixed for one minute with the water agar in each test tube using a Vortex Jr. mixer. The coated seeds were then placed on the seeded water agar in the test.tubes. The tubes were placed on a test tube rack slanted at a 60° angle. and incubated under continuous fluorescent light (8,600 lux) at 25°C. Uncoated surface-sterilized seeds sown on seeded and unseeded water agar were used for comparison. Disease development was observed as the roots grew into the 'agar. and disease was assessed ten days after planting. Disease sever- ‘ity was rated on a scale of 0-3; 0 being disease free. 1 being slightly diseased with 1-10% of the root system infected. 2 being moderately diseased with 11-25% of the root system infected, and 3 being severely diseased with more than 25% of the root system infected. RESULTS Effect of soil temperature and inoculum concentration on disease severity Soybeans (cultivar 'Harosoy'63') were grown in soil infested with different inoculum concentrations from 0.31 g to 5 g of soybean- sand culture/kg soil and incubated in 16°C. 20°C, 24°C, and 28°C water tanks. After three weeks disease severity was rated on a scale from 0-5. 0 being disease-free and 5 being most severely diseased. Disease severity decreased markedly as the temperature in- creased, but less markedly with decrease in inoculum concentrations (Figure 2). At 16°C each inoculum concentration used produced at least four times as severe a disease index as did corresponding inoculum concentrations at 28°C. At all temperatures tested. except at 28°C. disease severity at 0.31 g inoculum/kg soil was at least half that at 5 g inoculum/kg soil. .At 28°C, 5 g inoculum/kg soil was required to ' produce as severe a disease index as 0.31 g inoculum/kg soil at 16°C. Results obtained by Chen and Lockwood (unpublished results) on the effect of soil temperatures on disease severity of soybeans caused by I, basicola were thus confirmed. These temperature effects provided important clues for the biological control of the disease: a) if anta- gonists were to be used. they should be able to grow well and perform antagonism at lower temperatures where conditions for disease develop- ment are suitable; b) protection against the pathogen may be needed only during the early period of the growing season when soil tempera- tures are in the range favorable for disease development. 24 25 SI 1 I 4.. I I .2 I 3.. § _ s 1 I .3 (D 21 g 1 1 m '5 1' I 1 I . I 0.. I 1' / k- / / / /. / // 2/____./_/__-_- / / / / / / / / / // / 16°C 20°C 24°C 28°C Soil temperature Figure 2. Effect of different soil temperatures and inoculum concentrations on root disease severity of soybean cultivar 'Harosoy- 63' caused by Thielaviopsis basicola. Each bar represents the mean of four replicates with 4-5 measurements in each replicate. 26 Collection of antagonists Seventeen bacteria, forty-six fungi and five actinomycetes which produced inhibition zones against I, basicola or overgrew the pathogen were isolated by the bilayer method from rhizospheres of soy- beans grown in soils with different amendments. Of the forty-six fungal isolates. twenty-two belonged to the genus Trichoderma (8), which were then grouped into species aggregates (64): I, harzianum (thirteen isolates), I, pelluliferum (six isolates). and I, hamatum (three iso- lates). Based on the Saccardo system of classification (8). except for two asporogenous deuteromycetes. the other twenty-two isolates were grouped into the following genera: Penicillium, Aspergillus. Glioclad- Igm, Fusarium. Alternaria, Myrothecium. Rhizopus, and Actinomucor. The actinomycetes were all of the genus Streptomyces. Bacterial isolates A were not identified. I Using the paired culture technique, four bacteria. two fungi (both of the genus Fusarium). and five actinomycetes (Streptomyces spp.) from the rhizospheres of soybeans grown in the greenhouse, and eight actinomycetes (Straptomyces spp.) from the Fusarium mycelial mats showed antagonism to I, basicola either through the production of in- hibition zones or overgrowth. Isolates obtained by the bilayer method are designated by the prefixes ’b' (bacteria). 'T' (Trichoderma spp.). 'f' (fungi other than Trichoderma), and 'a' (actinomycetes). Those obtained directly from the rhizosphere are designated by the prefixes 'sb' (bacteria). 'bf' (fungi). and 'sa' (actinomycetes); and those obtained from the Fusarium 27 mycelial mats are designated by 'L' (actinomycetes). After puri- fication through successive transferring. the antagonists were preserved. Analysis of antagonism on agar.plgtes 1. Bacterial isolates. Spraying a suspension of I, basicola endoconi- dia onto individual bacterial colonies grown on PDA or seed exudate agar in most cases resulted in the production of inhibition zones. All twenty-one isolates produced inhibition zones on PDA although three of them were indistinct. Seventeen isolates also produced inhibition zones 'on seed exudate agar but two of them were indistinct. Two isolates with distinct inhibition zones and two of the three with indistinct ones on PDA produced none on seed exudate agar. Six other isolates produced weaker inhibition zones on seed exudate agar than on PDA. MiCroscopic observations were made around the inhibition zones on PDA one, two and three days after application of I, basicola endoconidia. Production of (distinct or indistinct) inhibition zones 'by nineteen isolates was associated with poor germination, twelve pro- duced inhibition zones associated with bursting of spores or spores in chains. and all twenty-one isolates lysed hyphae and endoconidia. Nine isolates showed a spreading growth habit both on seed exudate agar and on PDA (Table l). Agar discs or squares transferred from the inhibition zones produced on seed exudate agar to fresh PDA indicated that eight of twenty-one isolates produced diffusible antibiotics. Production of larger inhibition zones on PDA or seed exudate agar seemed correlated 28 .mwuwcooovcm can mega»; mo mwmxgu .emmm mpmnzxm town uemmno owadumoeowz . . . cowumnwzcu .owuwcououcw opouwmon mwmnmw>mpmwgh mo commcwamzm m cpwz mmpmpomw pmzuw>wvcw ucwcwmpcou mmympn Lame mcwxugam emumm made omega mmumpomw Fewemuuma mo umnm; gpzoem ucm .xppmuwaoumoeows umcpeewwwu mm cowu_nw;:_ us» we mu_ummeouuweoco .:o_pu:uoea u_uownwpcm .mmcoN cowuwnwccw ea cowuoavoea ._ mPQMH 29 with antibiotic production (Tables 1 and 2). I, basicola endoconidia on agar discs or squares from the newly formed inhibition zones showed low viability when transferred to fresh PDA. Only two of the eight agar pieces transferred showed growth of I, basicola during a one-week's incubation period (Table 2). Bursting of spores appeared to correlate positively with loss of viability (Tables 1 and 2). Seeds coated with bacterial antagonists were placed on carrot- extract agar plates (prepared by adding 1.5% Difco agar to the carrot- extract broth) seeded with 106 I, basicola endoconidia/ml. to determine the effect of inoculum from coated seeds on growth of the antagonist ‘colonies and on inhibition zone formation. Coating was done by soaking surface-sterilized (fifteen minutes in 1000 ppm NaOCl) soybean seeds in individual cell suspensions for one minute. Comparable plates were prepared similarly, but the seeds were removed from the plates after one minute. Plates treated with seeds soaked in sterile distilled water were also prepared as the control. The plates were incubated at 25°C under continuous fluorescent light (8.600 lux) for five days before diameters of inhibition zones and of bacterial growth were measured. Each treatment consisted of two to three replications. Growth of the bacterial colonies and production of inhibition zones were more prominent on agar plates with the coated seeds after five days of incubation; the diameters of growth plus inhibition zones were wider by 7-53 mm than those where the seeds were removed (Table 3). The diameters of growth alone (excluding the inhibition zones) were wider by 5-32.7 mm. One isolate (sb3) even failed to grow on the seeded agar in the absence of the seeds. 3O .m—oo_mmn .h mo cuzoem o: n 1 memmao mm: upcommma .h we cpzocw u + n .muwz Eu P A mean corpwawsc? u +++ ”mew: Eu o.F1m.o mco~ cowpvnvgca u ++e - ++ . “Fe - ++ mpg 1 +++ an 1 +++ up; 1 ++ on + ++ mg + ++ an - _ ++ Ne n»u_FwaeP> eocou eowuweweeH «beFOmH .mmcoN cowuwnwzcw.vmu=uogawg as» cm mpvwcoooucm mpouwmma mwmmow 1>apaweh to Agw_wnew> ecu .eoebms eaaepwn 8;» zn eaeweuao mmpeFOmv Feweapuee ago to mmcoN comuwnwgcw Eogm :mxmu Lama mpmvaxm.ummm,>n mmcoN cowpwawgcw mo,cowpu=voeqmm .N m—amh 31 .m:o_mcmam:m —_wo pewcmuumn eo ummumcw Lopez umppwumwc mpwcmum cw umxmom mummmu .mcowpmuPFame «menu on oz» mo mcmmza .oemsamonce map sag» appuwgwu .mmxwmmea .nm. saw: omega use ”voguos emxu_wn asp an uwcwwpno mew: mmxwmmea .n. saw: mmumpomfie o o o o o_oeucou N.mm n.mm o o mam N.mm «.mm . o.mp o.m~ Nam o.ou m.ov n.m¢ ~.NN N—n o.mn m.m¢ o.mm m.P¢ an mcoN coppwn mwgmpumn mco~ cowuwn ewemuomn 125:? + epzoea to cpzoea 125;? + £83028 .r to £82028 epmwcomepce um>oeme no: ummm um>oeog ummm Fewempumm gassv LmumEmwo .muacws mco Lmymo um>osme ago: so .mzwu w>we Low comm esp co new, emguwm mew: mvmmm .mwuwcououcw m_ouwmmn mwmaww>mpmwch saw; umemmm some womcpxm1pocemu ob mummm 28w: umwpqam mpmwcommpcm meempomn mo :pzoem use cowpuzuoca mcoN coppwnwccH .m mpnmh 32 The results showed that the presence of the seeds enhanced growth of antagonists and resulted in formation of wider inhibition zones, although variation among treatments occurred. This phenomenon was no doubt due to seed exudate and it suggested that an important consideration in the selection of antagonists might be their ability to make use of the substances exuded from the seeds and possibly roots. 2. Actinomycetes. Inhibition zones produced by this group of micro- organisms were also larger on PDA than on seed exudate agar. The inhi- bition zones were also associated with poor germination, bursting of spores, and lysis of hyphae or endoconidia. Of the eighteen inhibitory isolates. poor germination was associated with thirteen. lysis with nine. and bursting of spores with four (Table 4). Tests for antibiotic production in seed exudate broth showed that only three of the eighteen isolates produced inhibition zones when filter paper discs impregnated with culture filtrates placed on PDA were sprayed with a suspension of I, basicola endoconidia (Table 4). Culture filtrates of two additional isolates inhibited growth of I, basicola on the filter paper discs. but no zones were produced. Agar squares containing I, basicola endoconidia transferred from the inhibition zones of the three isolates to fresh PDA showed loss of viability associated with two isolates (Table 5). Loss of viability in this assay did not involve bursting of endoconidia. 3. Egggi, All fungal isolates collected overgrew I, basicola in paired cultures. Six slower growing fungi (Penicillium sp.. Myrothecium sp.. and Gliocladium sp.) produced expanding inhibition zones into which the antagonists continued to grow. Two of these isolates (both of the 33 .mwvwcouoccm ecu mega»; yo mmng 8 .emmm mumuzxm vmmm "gmmao ownaumoL0wz w» wnwpc< cowpmnwscH .mwu_coooucm myoowmmn mwmaow>mpmwgh mo,:owmamam:m m Suva mmpm_omw szuw>vucv mcwcwop 1coo mmpm_a Lama mcwamegm empmm mace owes» AFPmUPQoumoeuws uwcweemymu mm cowpwnwncw.mnu mo muwumwewuomemcu new .:o_uo:uoca owuowawucm .mmpmozsocwpum An mmcoN cowgwnwccw.mo cowuosnoea .¢ mfinm» 34 1. .oopmou uo: mo: _om oco No monopom_ spw3.oopowoommo opoowmoo .» yo zuwpwoow> mopoowmoo .p wo cuzocm o£I.H1 meomoo mo: opoowmoo .H mo coxoeu u +o .oowz so o.F1o.o ocon cowuwowgcw u ++ “comm co ocoN cowpwowgzw o:.u:o .mom_o co zuzogm opoowmoo .h oz u A o .ogogomoNP52 on» Eoem AFHoocwo .moxpwogo .mm. osu ;p_3 wasp oco ”mums Hop—ooxe aneomau ocp Eogm .moxwmoeo .4. cow: omocu mooguos eozopwo mop >5 nonwouoo oeoz moxwmoeo .o. cow: monopomHo H Pom + . ++. m4 - I S 1 ++ mm H mm 85:32.. confine. e328. .momwo-goooo eoupwo any morocooeesm mocon cowpwowccw on» cw omowcooooco opoommoo mmmmow>opowgw to zuw—woow> oco .mouopomw ouoozsocwuoo to mooMLQFWm menopao sow: oopocmocosw momwo so on Loppwe xo moco~ cowpwowcow mo cowpooooeo .m opoop 35 genus Gliocladium) were observed to parasitize I, basicola. Again, the inhibition zones were more prominent on PDA than on seed exudate agar. and all were associated with lysis of hyphae and endoconidia of I, basicola. Bursting of endoconidia was associated with inhibition zones produced by four. and poor endoconidial germination was associated with inhibition zones produced by five of these six isolates (Table 6). Diffusible antibiotics were produced by eleven of twenty- seven isolates growing in seed exudate broth (Table 7). Trichoderma pelluliferum (T21) and I, hamatum (T31)~produced strong inhibition zones when filter paper discs were impregnated with their culture fil- trates. while I, harzianum (T11)‘produced none. Paired cultures of I, basicola and individual fungal isolates were prepared to test the viability of I, basicola in the overgrown areas. Homogenates of agar discs taken from the overgrown areas showed variation in viability, as indicated by the numbers of I, basicola colonies arising on the selective medium (Figure 3, Table 7). Homo- genates of agar discs taken from I, basicola monocultures (control plates) yielded an average of seven colonies of the fungus. Of twenty- seven fungal isolates. sixteen caused complete loss of viability of the overgrown I, basicola. Of these sixteen isolates. five produced indis- tinct inhibition zones while two failed to produce inhibition zones. One isolate which produced a strong inhibition zone yielded sixteen I, basicola colonies, a number much higher than that of the control. Pro- duction of antibiotics in culture. or size of inhibition zones. there- fore. could not be related to loss of viability. 36 1 ++ moo_z so m.O1~.o ocoN cowp_owz:w u + ”pocwumwo no: ocoN cowuwowzcw u A monoN :owppowzcw oz .o_owcoooo=o one mega»: mo mwmzzo .Lomo opoozxo ooom u eomno omnoomoeopz . . . :owuwowzcz .owomcooooco opoowmon m_mno_>o_owzh mo commcoomzm o zpwz mouopomw Posow>wocw mcwcwopcoo moon—o some mcwzoeom swoon ozoo ooezo appoowooomoco_a oocwsgopoo mo cowpwawzcw oz» eo mowpm_eopoooozo one .:o_p 1o=ooeo owpowowuco .oozpos LozoFPa oz» mo oopopom_ Posse an mocoN cowuwowzcw mo compozooeo .o ozone 37 Table 7. Production of inhibition zones by filter paper discs impreg- nated with culture filtrates of fungal isolates. and viability of Thielaviopsis basicola in fifteen- -day- -old overgrown cultures. Inhibition I, basicola a Isolates zonesb coloniesc Control f1 (Myrothecium sp.) f2 (Myrothecium sp.) f3 (Asporogenous deuteromycete) f4 (Penicillium sp. ) f5 (Fusarium sp. ) f6 (Penicillium sp.) f7 (FUsarium sp.) f8 (Gliocladium sp.) f9 (Penicillium sp.) O + +1+ + + f10 (Fusarium sp. fll (Fusarium sp. f12 (Aspergillus ) ) .sp.) fl3 (Alternaria sp.) f15 Gliocladium f16 Fusarium sp. f17 usar1um sp. fl9 Rhizopus sp ct1nomucor f20 f18 IAct1nomucor ( ( f21 Penicillium f22 (Aspergillus bfl (Fusarium sp. bf2 (Fusarium sp. T11 (Trichoderma T2 (Trichoderma fl4 IAsporogenous deuteromycete) sp.) 1 .) §P sp.) e11 ) harzianum) pelluliferum) hamatum)‘ I+|+I+l+ +Il+ll+ + llll++ Illl 11' T3] (Trichoderma. aIsolates with prefixes 'f' and 'T' were obtained by the bilayer method and those with 'bf' prefixes, directly from the rhizosphere. b- = N0 inhibition zone; i = no growth of I. basicola on discs. but no inhibition zone produced; + = inhibition zone 5. l- 1.0 cm wide;H inhibition zone >:l cm wide. CMean number of colonies of two plates; homogenates of agar discs taken from the overgrown areas were plated on a medium selective for I, basicola. 38 Figure 3. Viability of overgrown Thielaviopsis basicola in paired cultures with six different fungal antagonists as shown by the number of colonies arising on a selective medium. A) Rhizo us sp.. B) an asporogenous deuteromycete, C & D) Fusarium spp.. E & F Pen1cillium spp. Darkest colonies are I, basicola. some of which are indicated by arrows. 39 Antibiotic production and nutrient deprivation on 111111th Isolates known to produce inhibition zones through antibiosis and nutrient deprivation were individually streaked on PDA plates pre- pared at various dilutions from 1/1 to 1/128. After 3-4 days of incuba- tion. agar discs were taken from the margins of the antibiotic producing colonies and were transferred to fresh undiluted PDA plates. Control discs were taken from PDA with similar dilutions. Each treatment was done in two replications. The plates with agar discs and those with nutrient-depriving colonies were sprayed with a suspension of I, basicola endoconidia. Three days after spraying, inhibition zones were measured from the margins of the agar discs or colonies to the margins of the inhibition zones. Production of diffusible antibiotics increased with increase in nutrient content of the PDA. as shown by the widths of the inhibition zones produced (Figure 4). In some cases increase in dilution resulted to some extent in a slight increase in widths of inhibition zones pro- duced by the nutrient-depriving colonies (Table 8). but this phenomenon was not reproducible in repeated experiments. In other cases the more diluted the medium the narrower were the inhibition zones produced. Biological control experiments with saybean root rot caused by T. basicola Sieved. steamed soil mix was infested with a suspension of I, basicola endoconidia and chlamydospores and the final inoculum 4O .moozsopooepm o no: —4 one .Asswppwo_cozv mamas; o was me .owgooooo mom: on oco No .ooozoogo mocon cowpwowzcw ozu.oo snow: one an commoeoxo mo .mompownwuco opowmammmo yo cowuooooeo :o mo uoommu .o weamwm meo.o=_.o «a. 8/1 Z/l 8/1 2/1 I... / 8 8/1 2/1 Z/l 8/l Z/l BZL/l VQ/L ZE/l BZl/l V9/l ZE/l 8ZL/l 99/l ZE/l BZl/l 79/1 ZE/l BZl/l VQ/l ZC/l Foepcoo (ma) sauoz "0111919"! JO HlPlM 41 .mpcosooomoos oz» 4o mcoozo .moos pompous; Eswoomau oz» zoom .xwooeo .4. ozp cow: oozo oco oozpoe co>o_w4 oz» >4 oocwopoo oooz moxmmoeo .o. zuwz mouoPomHo m.m 3 mg a... .3 m... 5 o.~ 9N 9N _ 0; mo 3 me 9m 3 9m 9m 3.. 3 Ne wmp\. oo\. NM\_ o.\_ o\_ .\_ <9; oopapwo co AAEEV moamm:omownwz=m oo zpowz omopopomH {II 1 1 1 I1 11 III 1 ll .mopoozsocwooo mo mowoopoo mow>woooo1ucowoosc an opoowmoo mwmoow>oFowzh omcwomo mocoN cowuwowzcw mo :o_posooo co mo uoommu .w oFQMH 42 concentration was adjusted to 103-104 propagules per g of soil. This soil was used in the first four experiments. In the first experiment ten bacterial and eighteen fungal isolates were tested. each with 9-12 single plant replications. One bacterial isolate (sb2) showed an average disease index of 1.8. as compared to 2.9 for the control (Ef0.05) (Figure 5. Table 9). In the second experiment. nine bacterial and thirteen fungal isolates were tested. each with 7-10 single plant replications. One bacterial isolate (b9) showed an average disease index of 2.3. as com- pared to 4.0 for the control (E?0.01); another bacterial isolate (sb4) significantly reduced disease index to 2.5 (P?0.05) (Figure 6. Table 10). In the third and fourth experiments. nineteen actinomycete and four fungal isolates were tested. each with 6-10 single plant replications. Four actinomycete isolates (sa4. sa5. L8. and a3) showed average disease indices of 1.9. 2.1. 2.1. and 2.1, respectively. as compared to 3.9 for the control (Ef0.01) (Table ll). One other actino- mycete isolate (sa2) significantly reduced disease index to 2.4 (E§0.05). Three fungal isolates (f20. f21. and f22) gave average disease indices of 1.9. 2.0. and 2.4. respectively, as compared to 4.5 for the control (£f0.01) (Table 11). One fungal isolate (bf2) significantly reduced disease index to 3.2 (Ef0.05). Sieved Conover loam-white silica sand mix was infested as previously described. but the final concentration was adjusted to 104- 105 propagules per g of soil. Three bacterial. two actinomycete. and three fungal isolates which had reduced disease severity significantly in the previous experiments were tested using this soil. In addition. 43 Figure 5. Reduction in severity of soybean root rot caused by Thielaviopsis basicola. by antagonists applied to the seeds. The seeds were sown in steamed soil mix infested with the pathogen. Left. control; center. seed-coating with bacterial isolate sb2; right. seed-coating with fungal isolate Tl3 (Trichoderma harzianum). Figure 6. Reduction in severity of soybean root rot caused by Thielaviopsis.basicola, by antagonists applied-to the seeds. The seeds were sown in steamed soil mix infested with the pathogen. Left, control; center. seed-coating with bacterial isolate b9; right. seed- coating with bacterial isolate sb4. 44 45 Table 9. Effect of bacterial and"funga1 antagonists applied to soybean seeds on root rot caused by Thielavigpsis basicola. Seeds were sown in a previously steamed soil mix infested with endoconidia and.chlamydospores of the pathogen. Antagonistsa Disease Indexb Control A 2.9 Bacteria 3.3 b2 2.7 b3 2.4 b4 3.0 b5 2.9 b7 3.8 b8 2.5 sbl 2.1 sb2 l.8* sb3 2.1 Fun i TI (Myrothecium sp.) f3 Asporogenous deuteromycete) Penicillium sp.) . f5 IFusarium sp.) Penicillium sp.) f7 (Fusarium sp.) f8 (Gliocladium sp.) f9 (Renicillium sp.) f10 (FUsarium sp.) bfl (Fusarium sp.) T11 (Trichoderma-harzianumI tl2 (TriEhoderma- arz1anum 3 ‘(Trichoderma harzianfim) T14 (Trichoderma harzianum. 5 (Trichoderma harzianum) (Trichoderma harzianum) T21 (Trichoderma pelluliferum) T33 (Trichoderma-hamatum) Shortest Significant Range (SSR)C, Ef0.05 N N wmwmmw NNOONN NNNNOO O O O O O O O O O O O O 0 oo '-' #mN-P-PO onto—1004:- GNQQN aEach treatment had 9-12 single plant replications; isolates with pre- fixes 'b', 'f'. and 'T' were obtained by the bilayer method; and those with prefixes 'sb' and 'bf'. directly from the rhizosphere. bDisease was estimated on a scale of 0-6. with 0 indicating no disease and 6. severe disease. cDuncan's Multiple Range test; the disease index for each isolate was compared with that of the control only. 46 Table 10. Effect of bacterial and fungal antagonists applied to soy- bean seeds on root rot caused by Thielaviopais basicola. Seeds were sown in a previously steamed—soil mix infested with endoconidia and chlamydospores of the pathogen. —v— b Antagonistsa ' Disease Index h 0 Control Bacteria 5 b9 blO bll b12 b13 b15 b16 b17 Fun i fll (Fusarium sp.) f12 (Aapergillus sp.) fl3 (Alternaria sp.) fl4 (AEporogenous deuteromycete) f15 (Gliocladium sp.) f16 IFusarium sp.I ii- 1- . C O HMGDN 00001001901 (IO-FOO“ NNOONN O O fl7 Fusarium sp. f18 Actinomucor sp.) f19 (Rhizopus sp.) Tl5 (Trichoderma-harzianum) T17 (Trichoderma harzianum) T22 (Trichoderma pelluliferum) T34 ([richgderma hamatum) Shortest Significant Range (SSR)C1 0100. 05de 0010‘ \ILONN-h -'—" 00000000 awn)!» 0000000300 0 O O O O O O O O 0.05 0.01 P. P 'fi aEach treatment had 7-10 single plant replications; isolates with pre- fixes 'b', 'f'. and 'T' were obtained by the bilayer method; and that with the 'sb' prefix. directly from the rhizosphere. bDisease was estimated on a scale of 0-6. with 0 indicating no disease and 6. severe disease. cDuncan's Multiple Range test; the disease index for each isolate was compared with that of the control only. 47 Table 11. Effect of actinomycete and fungal antagonists applied to soybean seeds on root rot caused by Thielaviopsis basicola. Seeds were sown in a previously steamed soil mix infestedf with endoconidia and chlamydospores of the pathogen. f . a . b SSRC Antagon1sts Disease 1ndex E?0.05 £f0.01 Control 3.9 Actinomycetes L1 3.2 L2 2.7 L3 3.1 L4 4.4 L5 4.2 L7 3.3 L8 2.1** 1.7 a1 3.1 a2 3.4 a3 2.1** 1.7 a4 4.0 a5 3.2 sal 2.9 sa2 2.4* 1.3 sa31 3.6 sa32 3.8 sa4 2.1** 1.7 sa5 l.9** 1.9 Control 4.6 Fun i 20 Actinomucor sp.; 2.4** 1.5 f21 IFenicilIium sp. l.9** 1.9 f22 Aapergillus sp.) 2.0** 1.5 bf2 (FUsaFium sp.) 3.2* 1.2 aEach treatment had 6-10 single plant replications; isolates with pre- fixes 'a' and 'f' were obtained by the bilayer method; those with pre- fixes 'sa' and 'bf'. directly from the rhizosphere; and those with the 'L' prefixes. from the Fusarium mycelial mats. Disease was estimated on a scale of 0-6. with 0 indicating no disease and 6. severe disease. CShortest Significant Range from Duncan's Multiple Range test; the disease index for each isolate was compared with that of the control only. b 48 five bacterial and four fungal isolates which had produced lower disease indices (but not significantly). and one avirulent I, basicola isolate 173 (Maduewesi and Lockwood, unpublished results) were included. Each treatment consisted of 8-16 single plant replications. Of the eighteen isolates tested. plants from seeds coated with three bacterial isolates (sbl. sb2. and sb3) and one fungal isolate (Fusarium sp.) had disease indices of 1.5. 1.4. 1.6. and 1.6. respectively. as compared to 2.6 for the control (E?0.05) (Table 12). Although only four isolates showed statistically significant differences from the control. all except one (L8) produced disease indices lower than that of the control. The fungal isolate which gave significant biological control in Conover loam mix was identified as Fusarium galagi (29). The two bacterial isolates sb2 and sb3 were identified as Bacillus spp. and the bacterial isolate sbl was identified as Achromobacter sp. Several characteristics of those bacterial isolates are listed in Table 13. Effect of coated soybean seeds on root rot development in water agar Water agar in glass test tubes was seeded with I, basicola endoconidia and soybean seeds coated with individual antagonists were placed on the top of the agar columns. Uncoated seeds and unseeded water agar were used for comparison. After ten days' incubation under continuous fluorescent light (8.600 lux) at 25°C. root rot development was observed. Root rot severity was rated on a scale of 0-3. with 0 indicating no disease and 3, severe disease. Table 12. Effect of bacterial. actinomycete, and fungal antagonists applied to soybean seeds on root rot caused by Thielaviggsis basicola. Seeds-were sown in a Conover loam-white silica sand mix infested with endoconidia and chlamydospores of the pathogen. Antagonistsa Disease indexb SSRc P=0.05 Control 2.6 Bacteriad . sb 1.5* 0.9 sb2 1.4* 0.9 sb3 1.6* 0.9 sb4 2.5 b8 2.0 b9 2.5 bll 2.2 b12 1.9 Fun id Tfiielaviopsis basicola iso ate f17 IFusarium sp.) f20 Actinomucor sp.) f21 IPeniciIlium sp.) f22 Aspergillus sp.) bfl (Ffisarium sp.) Tl2 (Trichoderma harzianum) T21 (TriEhoderma pelluliferum) Control Actinomycetese a3 L8 N N—‘d-HNN-JN NM 0 O GIN aIsolates with prefixes 'b', 'f'. 'T', and 'a' were obtained by the bilayer method; those with prefixes 'sb' and 'bf'. directly from the- rhizosphere; and that with the 'L' prefix. from the Fusarium mycelial mats. bDisease was estimated on a scale of 0-6. with 0 indicating no disease and 6, severe disease. CShortest Significant Range from Duncan's Multiple Range test; the disease index of each isolate was compared with that of the control only. d Each treatment had 9-16 single plant replications. 6Each treatment had 8-12 single plant replications. 50 Table 13. Several morphological and physiological characteristics of three bacterial antagonists giving significant biological control of soybean root rot caused by Thielaviopsis basicola in Conover loam. . . Isolates Character1st1cs 951 $52 553 Gram stain Negative Positive Positive Shape Small rods Rods Rods in chains Size (microns) 0.5 x 0.7 2.7 x 0.6 8.0 x 1.5 Motility Motile Motile Motile Endospore production Negative Positive Positive Litmus milk reaction Non-acidic Non-acidic Non-acidic No clearing No clearing Clearing Oxygen requirement Obligate Obligate Obligate aerobe aerobe aerobe H25 test Negative Negative Negative Reaction in phenol- Slightly Basic Basic red dextrose agar basic Fluorescence in Negative Negative Negative King's 8 medium Catalase test Weakly Positive Positive positive 51 One bacterial antagonist (b9) significantly reduced root rot severity with an average disease index of 1.7. as compared to 2.7 for the control (Ef0.05) (Figure 7. Table 13). Two fungal antagonists (bfl and T13) also significantly reduced root rot severity with disease indices of 1.7 and 0.2 (£30.05and~330.01. respectively) (Figure 7. Table 14). The avirulent I, basicola isolate 173 again failed to reduce root rot severity significantly. One bacterial (sb2) and one fungal (f9) antagonists gave disease indices higher than that of the control. 52 Figure 7. Reduction in severity of soybean root rot caused by Thielaviopsis basicola. by anta- gonists applied to the seeds. ’The seeds were sown in water agar seeded with the pathogen. Left. control; center. seed-coating with bacterial isolate b9; right. seed-coating with fungal isolate Tl3 (Trichoderma harzianum). ' 53 .zpco Fog»coo oz» mo »ez» z»»z oozeoEoo me: o»e»om» zoeo Loo xoo=» omeomwe oz» w»mo» omcem o—o»»P=z m.=eoc=o 5oz» omcem »ceo»»»=m»m »mo»eozmo .omeomwe ozo>om .m oze omeomwo o: m=»»eo»o=» o z»»z .m1o oo opeow e co eo»eew»mo me: omeomwoz .m»es pewpooze geezemem oz» zoom .xweooo .4. oz» z»»z »ez» one mozozomoNsz oz» zoom >F»ooo»e ..ez. eze .zm. mox»eoeo z»_z omoz» meoz»oe oozepwz oz» zo oocwe»zo ozoz .h. oce ..m. ..z. moxwoogo z»»z mo»e»om» mmzo»»eo»_ooz »:e»o opmcwm e1~ oez »coE»eoz» zoeme o.m A.om moozso»ooe»mv m4 o»oozsoc»»o< N.» e«N.o Asecepnzez eegomozope V th m.o em.» A. no Eapcemzmv Fez o.m . Adm Eezpwommwhr aw m.~ mm» o»e»om» epoopmez. emcee o.m New N.N Pom me . 1: me e»zo»oem N.N Foe»zoo .01... 861m. 5 ozmm zxoec» omeomwo em»mwzo e»c< . .eFoowmez m_wmo»>e_o»zp mo ewowcoooeco z»»3 eoeoom zeme eo»e3 :» »:osoopo>oe »oe »ooz co m»m»:ome»ze z»»3 eo»eoo mooom zeozzom oo »oooou .ep opze» DISCUSSION Broadbent et_aI, (14) found that of more than 3,500 bacterial and actinomycete isolates tested, about 40% inhibited one or more of nine root pathogens on agar and about 4% were effective in steamed soil; even fewer would be expected to be effective under commercial conditions. They chose non-rhizosphere soils as the sources of antagonists. Due to the very low probability of obtaining antagonists effective in field conditions. they suggested that large-scale screening methods are esSen- tial for success in biological control. Even if an introduced organism became established in a soil at a level of population higher than in the uninoculated soil. there is still no guarantee that it would be antagonistic to a pathogen on or near the root surface. because this might not be a suitable substrate for the introduced organism (36). One hundred percent of the seventeen bacteria and five acti- nomycetes isolated by the bilayer method were antagonistic to I, gagi- EQIa_in paired cultures. Antagonism was either associated with inhibition of endoconidial germination. bursting of endoconidia, lysing hyphae and endoconidia, or with a combination of the three mechanisms ’ (Tables 1 and 4). The success in biological control in steamed soil was also greater than that found by Broadbent et_aI, (14). Of these twenty-two isolates, one bacterium and one actinomycete (9.1%) signifi- cantly reduced root rot in steamed soil mix. 54 55 The bilayer method also was highly efficient in obtaining fungal antagonists. Of the forty-six fungi isolated. all overgrew I, basicola in paired cultures; sixteen of twenty-five isolates tested caused a complete loss of viability of the pathogen in overgrown areas. while an additional four caused a marked reduction in viability (Table 7). Of thirty-five isolates tested for biological control (Tables 9- 12), three (about 8%) significantly reduced root rot in steamed soil. On the other hand, antagonists obtained directly from the rhizosphere by random selection showed a higher proportion of success in the biological control experiments. Of the four bacteria so obtained, two (50%) gave significant biological control in steamed soil and three (75%) in Conover loam; one of these reduced disease significantly in both soils (Table 15). Of the six actinomycetes thus isolated, three (50%) gave significant biological control in steamed soil and none in Conover loam (Table 16). Of the two fungi thus isolated. one (50%) gave significant biological control in steamed soil and one (50%) in Conover loam. Overall, six of twelve isolates reduced disease in steamed soil. and four of twelve did so in Conover loam. Actinomycetes isolated from the lysed Fusarium hyphae resulted in 14.3% success in steamed soil (Table 16). Success in biological control was not closely related to anti- biotic production on agar plates or in liquid medium. Of the twelve antagonists significantly reducing disease severity in steamed soil (Tables 15-17). only three produced antibiotics and these three anta- gonists failed to reduce disease severity significantly in Conover loam. Only one of eight bacteria. one of three actinomycetes. and one of ten 56 Table 15. Results of laboratory observation of bacterial antagonists as related to their performance in the b1ological control experiments with soybean root rot caused by Thielaviopsis basicola. w Disease indexe 1616 1: 2545 E x D .0 =1! u-co >,-1- .3... :0 U: U 0: 0':- .EC GJX OE ‘5 O: ""0 >5 "-0 U o m-:— 1— m "—0 4.9-.— JJ 44'!- Uhr" ”-0 :5 OJ “N 04-3 '1- (6+3 C: OO O I'— S— r— +J 0:- 01-0 I— :01- 'r-O U .C O 0 O «3 .D a: '1- 'f’-D +30 :11: C: S- > L. r— 'l- 'P'U .0 Eu- mo *I-QJ OJ 44 O +3 8 ‘E 28 .53 cu'g ‘53 £1. 23 5 S 5 1—1 r—c <9. > L'J-r- mm .40 (D U Q L.) b1 - - + + + 3.3 2.9 b2 +++. +++ - + + + 2.7 b3 - + + + 2.4 b4 +++ ++ + + — + 3.0 b5 + - + - + 2.9 b6 + + - + b7 + - + - + 3.8 b8 ++ ++ + + - + 2.5 2.0 2.6 sbl i - - - + 2.1 1.5* sb2 + - +’ - + 1.8* 1.4* sb3 - - - - ~+ 2.1 1.6* sb4 + - - - + . 2.5* 4.0 2.5 b9 +++ ++ - + + + 2.3** 2.5 blO i - + + + 3.9 bll + - + + + 2.8 2.2 b12 +++ +++ - + + + 2.8 b13 +++ +++ - + + + 3.7 bl4 - - + + + b15 +++ ++ - + + + 3.9 b16 + - + + + 4.3 b17 +++ ++ - + + + 3.1 I aIsolates with 'b' prefixes were obtained by the bilayer method and those with 'sb' prefixes. directly from the rhizosphere. bBased on seed exudate agar; - = no inhibition zone. i = inhibition zone not distinct; + = inhibition zone 0.1-0.5 cm wide; ++'= inhibition zone 0.6-1.0 cm wide; +++ = inhibition zone > 1 cm wide. c_ = Loss in viability; + = I, basicola remained viable. dMicroscopic observation on PDA plates; + = respective mechanism pre- sent; - = respective mechanism absent. eScale from O to 6; 0 being disease free and 6 being most severely diseased. *Indicates statistical significance at £30.05; **indicates statistical significance at £30.01. 57 Table 16. Results of laboratory observation of actinomycete antagon- ists as related to their performance in the biological con- trol experiments with soybean root rot caused by Thielaviop- §j§_basicola. Disease indexe W (U M .C'I- 'U O-‘U E x a an to 41l— ‘U >9'r" Mor- cd) 0 1:: O 0: o-:- .c: '0) X OS (U 0 C ‘l— 0 >3 '1' O 1! O m'r- I— ” ..— o 4.)..— 4.) +3-1— U'PI- ‘4- U 3 E a) +JN 04-» -1-- «3+: :1: oo o 1— L 1— » w- ~1— u 1— c-u- --— o 'o .c o a: o c6 .0 .D 3 w- -I-.D «HO m: C S- > S— u—- '1- °I-"U .D E'l- m 0 01- GJ 0 H O +9 O .C «H 2 M .: LU U) 0.) C 1: c m C C -u- 0 C 3 C >,!- S- O O O H H < D. > (.501— m d) ._l 0 <5 0 U U L] ++ ++ - + - + 302 309 L3 1- u- + - " 30] L4 1‘ " " - " 404 L5 : - + + - 4.2 L7 + - + - -' 303 a] i- - + - + 30] a2 i i + - + 3.4 a3 + — + + + 2.1** 2.2 a4 i - + + + 4.0 a5 ++ ++ - + - - 3.2 sal + i + + + 2.9 sa2 i - - - - 2.4* sa31 i - - - - 3.6 Sa32 i - - - 3.8 . sa4 i - + — - 2,1** sa5 - - + - + 1.9** aIsolates with 'a' prefixes were obtained by the bilayer method; those with 'L' prefixes. from the lysed Fusarium hyphae; and those with 'sa' prefixes. directly from the rhizosp ere. bBased on seed exudate agar or culture filtrate with seed exudate broth as growth medium; - = no inhibition zone; i = inhibition zone not dis- tinct; + = inhibition zone O.l-O.5 cm wide; ++ = inhibition zone 0.6- 1.0 cm wide. c_ = Loss in viability; + = I, basicola remained viable. dMicroscopic observation on PDA plates; + = respective mechanism pre- sent; - = respective mechanism absent. eScale from O to 6; 0 being disease free and 6 being most severely diseased. *Indicates statistical significance at Ef0.05; **indicates statistical significance at E?0.01. lQpSlS l 1men Results of laboratory observation of fungal antagonists as related to their performance Table 17. ts w 1 control exper the biolog basicola. th soybean root rot caused by Thielav 1C3 1n Disease indexe 58 lousuoa xIw weol JBAOUOQ louauog xIw asnouuaaug eIpIuooopua J0 geqdfiu JO sgsxq eIpIu030pua J0 Butqsung "OlliqLHUI _Botaeutwuag pwsgqgseued afieiliqeih uotaonpoud q01101911uv IBUOZ qUOLlIQIHUI P59191051 2.6 1.6* 2.0 01 N NmeQVmF-POSMOI— . . . C . O . O O C O O (')NNNNNN(',NN¢')N + + + + + I + 1 + 1 + + + O OF-Or—ONOOOMOOC + i i 1+|+++II+I++ I I++ + . + +1 ++ OF- F-F-r- PMQ’WONWO‘PQ—g—Nm H—‘I—‘I—‘I—‘O-‘I-‘I-‘I—‘I—DFI—I— 59 - . .Fo.omm »e ooceowmwcmwm _eo»»m»»e»m mo»eo»e:»«« “mo.omm »e ooceowmwzmwm Feo»»m»»e»m mo»eo»ozHe .eomeom»e zpooo>om .o ece oozm omeomwo m=»»eo»o:» o z»»3 “m1o 5oz» opeomo .»comze Emmzezooe o>»»ooomoz u 1 m»:omoeo Emwcezoos o>»»ooomoz u + ”mo»e_o zomzo owooomoeowze 1. .Foz»coo oz» 2» mo_zopoo co>om o» eozeosoo we .meoze c3ozmzo>o oz» 5oz» mommwze epoowmez .h mo mowzopoo mo goosezo .oe»: so _ A ozoN zo»»»zwz=» u +++ moo»: so o.P1m.o ozoN co»»»z 1»z=» u ++ woe»: so m.O1».o ocoN zo»»»z»z=» u + m»o:»»m»o »oc ozoN :o»»»z»z=» u n mozoN co»»»z»z=» o: u 1 “sowoos z»3ozm me z»ozz o»ee=xo eoom z»»3 o»ez»F»» oo=»»:o Lo zeoe o»eo=xo eoom co oomemz .m opzeh »P=m=oo .moxweozo eze mosecoweocom o» oocozomoz Loo .meozsez.o»e—omme In . 1 \ F 1. 113 +++ . «on 1 mm» 1 FN» 1 cm» mp» wpm up» m_w + mpm «F» +++ n»; it 'I‘it -?NI\O$I\H'OI&DO Q'O‘QN" I ' . Q'Q'O‘l ‘0 ¢ '3 '3'“ m .OF-F—omOI—F— OFOO‘ m." +1+ +1 0 0 K00”) +1 'N F 1.1.. 6O fungi which clearly produced diffusible antibiotics on agar plates or liquid medium reduced disease significantly in steamed soil; while four of thirteen bacteria. four of thirteen actinomycetes. and four of nine fungi which did not produce antibiotics reduced disease signifi- cantly either in steamed soil or in Conover loam. Therefore. antagonism by virtue of nutrient competition may be more important than antibiotics in biological control of I, basicola. Other characteristics of anta- gonism. either individually or in combinations. were also not consis- tently associated with success in biological control (Tables 15-17). Success in biological control in steamed soil appeared to be somewhat greater with isolates producing distinct inhibition zones in paired cultures on seed exudate agar (29%) than with those producing indistinct or no zones (17%) (Tables 1, 4, 6, 15-17). However. success in natural Conover loam was about equal for the two groups, i.e.. 6% and 8%. respectively. Production of inhibition zones on a too-rich medium may not reflect antagonistic ability in a medium similar to root exudate. Isolates that produced inhibition zones on PDA often produced none or. smaller zones on seed exudate agar (Tables 1 and 4). However. of the twenty-nine isolates producing distinct inhibition zones on PDA. 21% reduced disease significantly in either steamed soil or Conover loam. whereas of those producing distinct inhibition zones on seed exudate agar, 24% did so (Tables 1. 4. 15. 16). Therefore. the expected advan- . tage of using seed exudate agar in the evaluation of antagonists was not clearly shown. 61 In the absence of a set of antagonistic characters clearly predicting success in biological control. selection of antagonists from soybean rhizospheres at present appears to be the best course. Rhizo- sphere organisms may have a better capability than non-rhizosphere ones to colonize roots. and may more quickly and effectively establish a fungistatic environment in the rhizosphere, where germination of spores of root-infecting fungi occurs (78). Failure of biological control by many antibiotic- and the non-antibiotic-producing antagonists in natural soil may be due to their sensitivity to inhibitory substances produced by indigenous micro- organisms, or to inability to compete successfully for substrate (28). Failure of the antibiotic-producing antagonists may also be due to microbial degradation of their antibiotics. Indigenous antagonists of I, basicola may also have been eliminated due to their sensitivity to the antibiotics produced by the introduced antagonists. resulting in a weaker overall antagonism to the pathogen (l3). Exhaustion of nutrient in the rhizosphere may also occur quickly, resulting in lack of substrate for the production of antibiotics. Moreover, isolation of antagonists from the rhizosphere does not necessarily assure that they are rhizosphere residents. unless the frequencies of their occur- rence there are high. The results of the experiments using soil mixes and those using water agar seeded with I, basicola endoconidia showed some cor- relation. For example, of the eight isolates tested in agar, three produced disease indices significantly lower than the control (Table 14); and of these. bacterial isolate b9 significantly reduced disease 62 in steamed soil and reduced disease. but not significantly. in Conover 10am. The reverse was true for fungal isolate bfl. Trichoderma iso- late T13. that significantly reduced disease severity in water agar. also reduced disease in steamed soil (Tables 9 and 14). However. two bacterial isolates (sb2 and sb3) which gave significant biological control in Conover loam (Table 12) failed to do so in water agar; one even gave a disease index higher than that of the control (Table 14). These two bacteria did not produce diffusible antibiotics (Table 1), and might not have been able to grow along the soybean roots in the water agar to maintain antagonism in the rhizosphere. The technique using seeded water agar should be further developed and evaluated as an alternative approach for screening anta- gonists for biological control. Its advantages are that the test period is short (ten days). and it provides a transparent medium making possible continued observations of disease development and possibly of the interactions of the antagonists with the pathogen in the rhizosphere. Promising results have been obtained in the biological control experiments, and further work toward field testing should be carried out. To increase the probability of obtaining antagonists applicable for biological control under commercial scale. further screening from soybean rhizospheres with the aim of selecting those with high popula-‘ tions and those showing ability to colonize the root surfaces should be done. Suitable combinations of antagonists and suitable food bases applied together with the antagonists may provide higher degree of antagonism in the rhizospheres. 63 Since pathogenic propagules of roots must germinate in the rhizosphere in order to cause infection. continued effort to find antagonists capable of rapidly colonizing the rhizosphere and depleting its nutrients. and possibly.at the same time producing inhibitory sub- stances, may lead to further success in biological control. LITERATURE CITED 10. 11. 12. LITERATURE CITED Adams. P. B. and G. C. Papavizas. 1968. Survival of root- infecting fungi in soil. X. 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